IntroductionNuPotential Proprietary Technology Platform
Differentiated cells are thought to be stably committed to their fate; however, there is evidence to indicate that dedifferentiation events can take place. Urodele amphibians and teleost fish can replace lost anatomical parts by a process of migration, dedifferentiation, proliferation and redifferentiation of epithelial cells in the wounded area. Functional reprogramming of differentiated cell nuclei also has been illustrated by the derivation of pluripotent embryonic stem cells (ESCs) and the live birth of cloned animals after nuclear transplantation into unfertilized eggs. Notably, Xenopus eggs can reprogram mammalian somatic nuclei to express the POU family member homeodomain transcription factor gene Oct4 by a process requiring DNA demethylation. DNA demethylation also occurs after fusion of mouse thymocytes with embryonic germ cells (EGCs) or ESCs. Similarly, fusion of neuronal progenitor cells or bone marrow derived cells with ESCs results in hybrids that express markers of pluripotency, contribute to chimeras and form teratomas. Similar observations resulted from fusing human fibroblasts with ESs. Fusion of embryonal carcinoma cells (ECCs) with T-lymphoma cells also promotes the formation of colonies expressing pluripotent cell transcripts from the lymphoma genome. Thus, components of pluripotent EG, ES or EC cells have the potential of eliciting reprogramming events in a somatic genome.
As an alternative to fusion, somatic nuclear function also may be altered using nuclear and cytoplasmic extracts, with the rationale that extracts provide the necessary regulatory components. Notably, extracts of regenerating newt limbs promote cell cycle reentry and downregulation of myogenic markers in differentiated myotubes. Furthermore, it has been shown that kidney epithelial 293T cells permeabilized with Streptolysin O (SLO) and briefly exposed to an extract of Jurkat T cells take on T cell properties, including growth in aggregates, chromatin remodeling, expression of T cell-specific genes and surface receptors, secretion of interleukin-2 and stimulation-dependent assembly of the interleukin-2 receptor. Similarly, lysates of cardiomyocytes or insulinoma cells elicit expression of cardiomyocyte or beta-cell markers in adipose stem cells and fibroblasts, and a pneumocyte extract was recently shown to induce differentiation of ESCs into a pneumocyte phenotype. 293T cells were also shown to express pluripotency markers such OCT4 and germ cell alkaline phosphatase (GCAP) and downregulate a kidney marker after coculture with extract of Xenopus eggs.
In addition, the production of live animals after somatic cell nuclear transfer indicates constituents present in the oocyte have somatic cell reprogramming capability. Despite these observations, evidence for induction of epigenetic reprogramming events in large numbers of cells by any approach is lacking.
NuPotential addresses this important limitation by inducing epigenetic reprogramming events in large numbers of cells by altering nutritional components of media that bathe all cells in culture and supplementing these alterations with pharmaceutical and/or iRNA treatments. These treatments result in epigenetic changes of cells in culture and renewed capacity to differentiate into multiple lineages suitable for a variety of applications. Characteristics of these dedifferentiated cells include the formation of aggregates, re-expression of pluripotency markers, induction to multiple lineages and improved blastocyst development after SCNT.